Analytic ultracentrifuge calibration and determination of lipoprotein-specific refractive increments

Abstract

Accurate quantification of the major classes and subfractions of human serum lipoproteins is an important analytical need in the characterization and evaluation of therapy of lipid and lipoprotein abnormalities. For calibrating the analytic ultracentrifuge (AnUC), we routinely use a Beckman calibration wedge cell with parallel scribed lines 1 cm apart. Such a cell gives a rectangular pattern in the schlieren diagram, which determines magnification and also provides an area corresponding to an invariant refractive increment. We have independently validated this wedge calibration cell using a special boundary-forming cell in which 1.174% sucrose is overlayered with distilled water. Comparing wedge cell area with extrapolated zero time boundary area refractive increment gives agreement to within less than 1%, corresponding to a refractive increment error of ±0.00002 Δn. Complete calibration for AnUC analysis of lipoproteins also requires accurate determination of the specific refractive increments (SRI) of the major lipoprotein classes, namely low density lipoprotein (LDL) and high density lipoprotein (HDL). These are measured in the density in which they are analyzed, i.e., 1.061 g/ml for LDL and 1.200 g/ml for HDL. Five fresh serum samples were fractionated for total LDL and total HDL and their SRI determined. Total lipoprotein mass was determined using precise CHN elemental analysis and compositional analyses. The results yielded corrected SRI of 0.00142 and 0.00135 Δn/g/100 ml for LDL and HDL. Thus, our current values using 0.00154 and 0.00149 Δn/g/100 ml underestimate LDL and HDL by 9% and 11%. Corrections of all previous LDL and HDL AnUC data can be made using appropriate factors of 1.087 and 1.106.